The methylotrophic yeasts including Pichia pastoris have been widely used for production of recombinant proteins of commercial or medical importance. However, production and medical applications of some therapeutic glycoproteins can be hampered by the differences in the protein-linked carbohydrate biosynthesis between these yeasts and the target organism such as a mammalian or human subject.
Protein N-glycosylation originates in the endoplasmic reficulum (ER), where an N-linked oligosaccharide (Glc3Man9GlcNAc2) assembled on dolichol (a lipid carrier intermediate) is transferred to the appropriate Asn of a nascent protein. This is an event common to all eukaryotic N-linked glycoproteins. The three glucose residues and one specific α-1,2-linked mannose residue are removed by specific glucosidases and an α-1,2-mannosidase in the ER, resulting in the core oligosaccharide structure, Man8GlcNAc2. The protein with this core sugar structure is transported to the Golgi apparatus where the sugar moiety undergoes various modifications. There are significant differences in the modifications of the sugar chain in the Golgi apparatus between yeast and higher eukaryotes.
In mammalian cells, the modification of the sugar chain proceeds via 3 different pathways depending on the protein moiety to which it is added. That is, (1) the core sugar chain does not change; (2) the core sugar chain is changed by the addition of the N-acetylglucosamine-1-phosphate moiety (GlcNAc-1-P) from UDP-N-acetyl glucosamine (UDP-GlcNAc) to the 6-position of mannose in the core sugar chain, followed by removal of the GlcNAc moiety to form an acidic sugar chain in the glycoprotein; or (3) the core sugar chain is first converted into Man5GlcNAc2 as a result of the removal of 3 mannose residues by mannosidase I; and Man5GlcNAc2 is further modified by the addition of GlcNAc and the removal of two more mannose residues, followed by the sequential addition of GlcNAc, galactose (Gal), and N-acetylneuraminic acid (also called sialic acid (NeuNAc)) to form various hybrid or complex sugar chains (R. Kornfeld and S. Kornfeld, Ann. Rev. Biochem. 54: 631-664, 1985; Chiba et al J. Biol. Chem. 273: 26298-26304, 1998).
In yeast, the Man8GlcNAc2 glycans are not trimmed. The modification of the sugar chain in the Golgi apparatus involves a series of additions of mannose residues by different mannosyltransferases (“outer chain” glycosylation). The structure of the outer chain glycosylation is specific to the organisms, typically with more than 50 mannose residues in S. cerevisiae, and most commonly with structures smaller than Man14GlcNAc2 in Pichia pastoris. This yeast-specific outer chain glycosylation of the high mannose type is also denoted as hyperglycosylation or hypermannosylation.
Glycosylation is crucial for correct folding, stability and bioactivity of proteins. In the human body, glycosylation is partially responsible for the pharmacokinetic properties of a protein, such as tissue distribution and clearance from the blood stream. In addition, glycan structures can be involved in antigenic responses. For example, the presence of α-galactose on glycoproteins is the main reason for the immune reaction against xenografts from pig (Chen et al., Curr Opin Chem Biol, 3(6):650-658, 1999), while the immune reaction against glycoproteins from yeast is mainly due to the presence of α-1,3-mannose, β-linked mannose and/or phosphate residues in either a phosphomono- or phosphodiester linkage (Ballou, C. E., Methods Enzymol, 185:440-470, 1990; Yip et al., Proc Natl Acad Sci USA, 91(7):2723-2727, 1994).
Hyperglycosylation is often undesirable since it leads to heterogeneity of a recombinant protein product in both carbohydrate composition and molecular weight, which may complicate purification of the protein. The specific activity (units/weight) of hyperglycosylated enzymes can be lowered by the increased portion of carbohydrate. In addition, the outer chain glycosylation is often strongly immunogenic which may be undesirable in a therapeutic application. Moreover, the large outer chain sugar can mask the immunogenic determinants of a therapeutic protein. For example, the influenza neuraminidase (NA) expressed in P. pastoris is glycosylated with N-glycans containing up to 30-40 mannose residues. The hyperglycosylated NA has a reduced immunogenicity in mice, as the variable and immunodominant surface loops on top of the NA molecule are masked by the N-glycans (Martinet et al. Eur J. Biochem. 247: 332-338, 1997).
Therefore, it is desirable to genetically engineer methylotrophic yeast strains which produce recombinant glycoproteins having carbohydrate structures that resemble mammalian (e.g., human) carbohydrate structures.